Gene Editing with TALEN®

TAL effector nucleases are customized DNA scissors that bind and cleave pre-selected DNA sequences. The first TALEN® was developed through a collaboration between the laboratories of Dr. Dan Voytas (CSO at Calyxt) and Dr. Adam Bogdanove at University of Minnesota and Iowa State University, respectively, in 2009. A functional TALEN® consists of a DNA binding domain, which is derived from transcription activator-like effector (TALE) proteins and a nuclease catalytic domain from a DNA nuclease, FokI. The DNA binding domain of TALE features an array of 33 or 34-amino acid repeats. Each repeat is highly conserved, with the exception of the so-called repeat variable di-residues (RVDs) at amino acid positions 12 and 13. The RVDs determine which nucleotide each repeat recognizes within the targeted DNA sequence. The simple one-to-one correspondence between TALE repeat binding to nucleotides in DNA sequences make it a straightforward process to assemble repeat arrays to recognize novel DNA sequences. There are no apparent limits to the targeting range of a customized TALEN®. At least one TALEN® target site can be identified in every 10-20 base pairs in any genome. Together, these features make TALEN® the most powerful tool in the current genome engineering field to date. Calyxt has implemented this nuclease engineering tool to produce the best-in-class plant genome modification platform.

Key Advantages of TALEN® Over Other Gene-Editing Technologies

In addition to TALEN®, we are aware of three other classes of nucleases that enable gene editing, including meganucleases, zinc finger nucleases and CRISPR/Cas9. Despite the availability of other gene-editing platforms, we currently rely on TALEN® because of the following benefits:

Intellectual property—We have a strong intellectual property position with respect to TALEN® technology and its use to make our product candidates. We have actively sought to protect our proprietary technologies and product candidates through the licensing of a portfolio of 81 issued patents and 170 pending patent applications.

Specificity—TALEN® may be designed to limit its DNA cleavage to the desired sequence and to avoid cutting elsewhere in the genome. This parameter is essential as plant genomes are highly complex; for example, the wheat genome comprises 17 billion bases.

Precision—It is possible to design a TALEN® that will cleave at any selected region in any gene. For example, there are four related FAD genes in the soybean genome. Our TALEN® edited only the two genes that produce fatty acids in the seed; no edits were introduced into the other related genes.

Efficiency—A large percentage of cells treated by TALEN® bear the desired gene edit. Because of TALEN® efficiency, only a handful of plants have to be regenerated to recover those with edits in our target gene. For example, three of 19 transgenic soybean lines expressing the FAD2 TALEN® transmitted heritable edits to the next generation.

Validation—We have a strong track record with respect to our technologies and expertise as we have successfully edited more than 20 unique genes in 6 plant species since our inception in 2010.

Ease of use—We have extensive expertise in the design and assembly of TALEN® and can generate thousands of TALEN® per week.